Project Summary Leukemia is the most common cancer in children, and although prognosis has improved over the last several decades, the mortality rate is still 15-20% in children diagnosed with acute lymphoblastic leukemia (ALL). Consequently, leukemia is a leading cause of disease-related deaths in children. Although new therapies have shown the potency of the immune system for leukemia elimination, much is still unknown regarding immune escape mechanisms in leukemia. Previous studies have implicated calcineurin (Cn), a serine threonine phosphatase, in the promotion of leukemogenesis in T-ALL, yet its role in B cell-ALL (B-ALL) has not been well established. Our lab discovered extended survival in immune-competent mice engrafted with leukemia in which the essential regulatory subunit, CnB was knocked down. Yet, survival was not as prolonged in Rag1-/- mice (lacking mature B and T cells; thus, no adaptive immunity) engrafted with calcineurin-deficient leukemia (shCnB). These data implicate Cn as an important mediator of immune evasion during leukemia progression. To identify downstream effectors of Cn-dependent immune evasion, we defined the cytokine profiles from control leukemia (shNS) and shCnB cells and found that IL-12 secretion in shCnB leukemia was significantly increased compared to shNS leukemia. IL-12 is an interleukin produced by antigen-presenting cells that induces interferon-gamma (IFN-?) production by both T and natural killer (NK) cells, resulting in cytolytic killing of cancer cells and other pathogens. To further explore this finding, WT and Rag1-/- mice were engrafted with B-ALL and administered recombinant IL-12 (rIL-12). Both WT and Rag1-/- exhibited a prolonged survival with rIL-12 administration. This points to a clear role of the innate immune system in immune surveillance of leukemia. NK cells have a major role in innate immunity thus I intend to test the hypothesis that IL-12 secretion is regulated by Cn in B-ALL cells and also potently activates NK cells resulting in leukemia clearance. In Aim 1, we will determine how Cn regulates IL-12 secretion in B- ALL cells by using pharmacologic and genetic methods to inhibit Cn to explore IL-12 secretion and also its downstream target NFAT in both human and murine B-ALL cells and also primary B-ALL patient samples. In Aim 2, we will define the contributions of NK cells to anti-leukemia immunity using models of NK deficiency and complementation assays. NK cell activation in response to shNS and shCnB leukemia cells will also be examined using cytotoxicity assays. Our goal is to better understand immune evasion in hematological malignancies to develop novel immunotherapies to control leukemia.